Abstract: Compared to other subtypes of breast cancers (BC), basal or triple negative (TN) BC suffers a poor prognosis, caused by limited understanding of the driver signaling pathways. Thus, for TNBC, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. PI's lab uses a research pipeline that utilizes transmitochondrial cybrid (cybrid) models. Cybrid system is an excellent cell model that allows comparing mitochondria from different cells (example: benign and TNBC cells with varying invasion/metastatic potential) in a common defined nuclear background. We apply multiple OMICs approaches in cybrid models to discover mitochondria-nuclear communication and mitochondrial energy reprogramming regulated cancer pathways. Using this research pipeline, we have recently published that metastatic TNBC has high energy-dependence to mitochondrial fatty acid ?-oxidation (FAO). We have also discovered that FAO is a critical regulator of Src oncopathway in TNBC. We validated the findings from cybrid models in parental BC cells, PDX models and clinical data. Proto-oncogene c-Src is one of the most commonly upregulated cancer pathways in TNBC. However, multiple clinical trials including our own trial showed only limited clinical benefit with single drug approach of Src inhibitors in unselected TNBC patients. Thus, it is important to understand the mechanism of activation and drug resistance of c-Src in TNBC to develop reliable treatment strategies to inhibit TNBC progression. N-myristoylation is a lipid modification with the attachment of a fatty acid, myristate, onto the N- terminal glycine residue of target proteins. Our preliminary analysis in cybrid models and parental cells suggest that FAO regulates myristoylation of c-Src by enhancing cytosolic availability of myristoyl CoA. In this project we will validate this interesting finding using systematic modulation of FAO pathway. Since frequent drug resistance occurs after Src inhibitor therapy in TNBC, we have also analyzed the potential drug resistance mechanisms for FAO or Src inhibitor therapy in TNBC. Our strong preliminary data suggest that drug resistance to FAO or Src inhibition is due to autophagy-mediated tumor survival that is regulated by the reactive oxygen species (ROS)-induced MEK/ERK pathway. Thus, this project will also validate this exciting mechanism using multiple research approaches. Considering our strong in vitro and in vivo preliminary data, we have proposed large-scale preclinical studies using multiple PDX TNBC models to determine benefit of combination drug strategy to overcome the resistance to FAO or Src inhibition therapy in TNBC. Overall, this proposal is highly significant as it is expected to 1) reveal the significance of mitochondrial crosstalk in the activation of Src signaling in TNBC and 2) develop strategies to repurpose the existing FDA approved Src targeting drugs with suitable combination therapy for rapid clinical translation to manage currently non-targetable TNBC.